Cotton is an important crop in many areas of the world, and biotechnology methods have been applied to produce cotton varieties with desirable traits. One such desirable trait is insect resistance. The expression of an insect resistance transgene in a cotton plant can confer the desirable trait of insect resistance. Many different factors influence the expression of a transgene, including the orientation and composition of the cassettes driving expression of the individual genes transferred to the plant chromosome, the chromosomal location of the transgene insert, and the genomic result of the transgene insertion. For example, there can be variation in the level and pattern of transgene expression among individual events that differ in the chromosomal insertion site of the transgene, but are otherwise identical. There can also be phenotypic and agronomic differences between events.
To make a transgenic cotton plant containing a single transformation event, a portion of a recombinant DNA construct is transferred into the genome of a cotton cell, and the cotton cell is subsequently grown into a plant. A cotton cell into which the event is initially transferred is regenerated to produce the R0 generation. The R0 plant and progeny plants from the R0 plant can be tested for any desired trait(s), but the effectiveness of the event can be impacted by cis and/or trans factors relative to the integration site in the transformation event. The phenotype conferred by the event can also be impacted by the size and design of the DNA construct, which can vary by the combination of genetic elements in an expression cassette, number of transgenes, number of expression cassettes, and configuration of such elements and such cassettes. Identifying an event with desirable traits can be further complicated by factors such as plant developmental, diurnal, temporal, or spatial patterns of transgene expression, or by extrinsic factors such as environmental plant growth conditions, water availability, nitrogen availability, heat, or stress. Thus, the ability to obtain an event conferring a desirable level of transgene expression and a desirable set of phenotypic and agronomic traits is not readily predictable.
Due to these numerous factors that have an effect on the efficacy of an event, it is necessary to produce and analyze a large number of individual plant cell transformation events in order to create an event having proper expression of the desirable trait and optimal phenotypic and agricultural characteristics suitable for commercial success. Creating a commercially valuable transgenic event requires extensive molecular characterization, as well as greenhouse and field trials with numerous experimental events over multiple years, in multiple locations, and under a variety of conditions. A significant amount of efficacy, phenotypic, and molecular data is collected, and the resulting data and observations are then analyzed by teams of scientists and agronomists with the goal of selecting one or more commercially suitable events. Such an event, once selected, is then used for introgression of the desirable transgenic trait into other genetic backgrounds using plant breeding methods, thus producing a number of different cotton crop varieties that contain the desirable trait and are suitably adapted to specific local agronomic conditions.
Transgenic cotton plants are known in the art, but only plants expressing Lepidopteran toxins or herbicide tolerance genes have been produced. There are no commercial transgenic cotton plants for the control of Hemipteran (such as lygus, cotton fleahopper and verde plant bug) and Thysanopteran (such as thrips) pests of cotton crops.
Lygus species can threaten a cotton crop from earliest squaring through cutout and final boll set. The insects pierce squares and damage anthers and other tissues. When squares are less than 5 millimeters long, they shrivel, turn brown, and drop from the plant. Damage to larger squares may be to anthers, styles, and stigma, and may interfere with fertilization. If many squares drop, the plant may put its energy resources into vegetative growth, resulting in tall, spindly plants with reduced yields. Lygus species also feed on and destroy terminal meristems, causing bushy plants. If lygus pierce the wall of young bolls (typically less than ten days old) and feed on young seeds, these seeds may fail to develop. Lint around the injured seeds is stained yellow, and may not mature normally.
Cotton fleahopper can cause excessive loss of cotton squares, resulting in reduced yield and harvest delays. Cotton fleahopper is a key insect pest of cotton in Texas and Oklahoma, and an occasional pest in New Mexico, Arkansas, Louisiana, and other mid-South states of the United States of America. When heavy populations of the insect pest are left uncontrolled, the yield loss can become extremely high. Cotton fleahopper nymphs and adults feed on the juices of tender plant parts, especially the terminal buds and small squares. Deformed or ragged leaves are often seen as a result of this feeding. The greatest damage is to small squares that are no larger than a pinhead. The small squares turn brown or black and shed after being fed upon. Heavily infested plants grow tall and whip-like, have restricted growth of fruiting branches, and usually produce only a few bolls near the top. The insect and its damage is thus hard to detect until economic losses have been sustained.
Verde plant bug, a native species, emerged as an important boll-feeder along the Gulf Coast of the United States of America during the past ten to fifteen years. During this time, cotton yields have suffered losses from cotton boll rot in areas of South Texas. Piercing-sucking insects feeding on cotton bolls have been implicated in introducing the bacterial disease that causes boll rot. Verde plant bug was the dominant boll-feeding sucking bug species (>98% of insects collected using a beat bucket) from peak to late bloom in cotton fields near the coast along the Coastal Bend of South Texas, from Port Lavaca to the Lower Rio Grande Valley in 2010 and 2011. It was common in fields within 8 km of coastal waters (average of 0.42 bugs per plant during peak to late bloom), while it was not detected in inland fields. Cotton boll rot was found on up to 25% of the open bolls inspected, the disease was concentrated in coastal fields where verde plant bug was found, and it was the major contributor to boll damage. Results from field surveys and verde plant bug feeding on caged plants supported the positive association of verde plant bug presence and subsequent harvest-relevant cotton boll rot in open bolls at harvest (Armstrong, J., Brewer, M., Parker, R., and Adamzyk, J. (2013) Verde Plant Bug (Hemiptera: Miridae) Feeding Injury to Cotton Bolls Characterized by Boll Age, Size, and Damage Ratings, J. Econ. Entomol. 106(1): 189D195). Verde plant bug feeding on cotton bolls also results in lint and seed staining.
Thrips have “punch and suck” mouthparts that allow them to punch a hole in a leaf cell, insert their maxillary stylets, and suck up the cellular fluids. When thrips feed on terminal buds, on tiny developing leaves and on fruiting structures, the injury can be severe. When thrips feed on young undeveloped leaves within the terminal bud, the resulting damage is magnified as those leaves develop and expand. This is because the damaged tissue fails to develop properly, while undamaged tissue continues to grow. After prolonged feeding or feeding by high numbers of thrips, seedlings have a ragged appearance, with visible silvery feeding sites on cotyledons and terminal leaf tissue. Over time, these silver areas will turn brown in color. Heavily injured leaves usually have a crinkled, tattered appearance and often curl upwards at the margins. Seedlings with this type of injury are often described as “possum-eared cotton.” Heavy thrips populations can stunt growth, cause death of the terminal bud (resulting in “crazy cotton”), delay fruiting, and reduce stand. Severe thrips injury can result in substantial cotton yield reductions. Both larvae and adults show a preference to feed on and in flowers, making them particularly difficult to control with chemical pesticides. In addition, Frankliniella occidentalis (Western flower thrips) is a very efficient vector of different plant topoviruses (e.g., tomato spotted wilt virus and Cotton leaf roll virus) that cause damage to cotton plants. In sum, thrips can severely impact early season stand and plant vigor and can be very costly to cotton farmer.
Chemical pesticides are the current insect control methods used against Lygus hesperus (Western tarnished plant bug), Lygus lineolaris (Tarnished plant bug), Pseudatomoscelis seriatus (Cotton fleahopper), Creontiades signatus Distant (Verde plant bug), and the thysanopteran pests Frankliniella spp and Sericothrips variabilis (Thrips). These chemical insecticide methodologies often require multiple applications and different types of chemical pesticides. For example, to control lygus as many as five to ten treatments per season may be required. To control thrips, two to three treatments per season may be required. At least one treatment is required to control cotton fleahopper.
The use of chemical pesticides to control insect pests increases the cost to the farmer growing cotton, particularly in regions experiencing high insect infestation, thus reducing any potential profit derived from cotton production. In addition, the use of different chemical pesticides with multiple applications can have a negative impact on the environment and beneficial insects. Further, the development of resistance to chemical pesticides has been observed in these cotton pests. For example, the western flower thrips, Frankliniella occidentalis Pergande, has shown resistance to a number of different chemical pesticides (Sten Jensen (2006) Insecticide resistance in the western flower thrips, Frankliniella occidentalis, Integrated Pest Management Reviews, 5: 131-146). Resistance to multiple classes of chemical pesticides (carbamate, organophosphate, and pyrethroid insecticides) used to control Tarnished plant bug (Lygus lineolaris) has also been observed (G L Snodgrass et al., (2009) Acephate resistance in populations of the tarnished plant bug (Heteroptera: Miridae) from the Mississippi River Delta. J Econ Entomol, 102(2): 699-707).
Because of the economic and environmental cost associated with the use of chemical pesticides to control insect pests of cotton, and the development of resistance to chemical pesticides, there is a need for a cotton plant that expresses an insecticidal toxin active against Lygus hesperus (Western tarnished plant bug), Lygus lineolaris (Tarnished plant bug), Pseudatomoscelis seriatus (Cotton fleahopper), Creontiades signatus (Distant) (Verde plant bug), and the thysanopteran pests Frankliniella spp and Sericothrips variabilis (Thrips).